The critical need for improved understanding of pathogenic factors important to mycobacterial infection, intracellular replication and induction of granulomatous disease, as well as the genes responsible for resistance to drugs, has grown acute with the worldwide epidemics of tuberculosis and HIV. Currently, the number of Mycobacterium tuberculosis infections has increased at an alarming rate due to the surge in HIV, drug use, inner city poverty, and homelessness. The emergence of multiple drug- resistant M. tuberculosis strains makes the need for improved control extremely urgent. Worldwide, M. tuberculosis infects >l billion people and is the leading cause of infectious disease death. This proposal aims to develop molecular tools, including differential display (dd)PCR, to study genes differentially expressed during M. tuberculosis infection, to facilitate rational vaccine development, and to devise clinically useful tests that can differentiate human hosts with active disease from those with dormant infection or distant past exposure. The same procedures for discovering differentially expressed genes would also be expected to be useful in detecting genes that are expressed in mycobacteria during distinctive growth phases, in response to specific culture conditions, nutrients, and in response to drugs. DdPCR and arbitrarily primed PCR of M. tuberculosis genomic DNA will also be used to identify newly acquired genes or genes altered through normal mutation which confer drug resistance to MDR-TB. Genes identified by ddPCR will be cloned and used in Northern blot analysis to establish which isolated cDNAs are preferentially expressed in virulent organisms. Whenever possible, the molecular techniques used to detect differential expression will be automated, taking advantage of new robotic workstations, fluorescent sequencers, and data processing systems recently installed at our facility. Gene products found to be expressed most abundantly in virulent organisms will then be analyzed phenotypically and in complementation assays. In particular, we will attempt to define surface antigens that may be differentially expressed that permit cell adherence and attachment. These proposed studies are designed to complement our ongoing research into M. tuberculosis virulence using more traditional techniques. Such multi-laboratory, multidisciplinary approaches should generate a better understanding of how M. tuberculosis invades and replicates in cells, evades host immune systems, cause granulomatous tissue destruction, and acquires drug resistance. This knowledge should allow us to define specific probes for the rapid diagnosis of drug resistance in clinical strains, and in future projects allow the re- designing of antibiotics. Virulence factors would be expected to be highly effective targets for vaccines, and to be useful in diagnostic tests aimed at identifying active disease.